This invention relates to biocompatible oxygen carriers for administration to patients as a supplement for or a partial replacement for whole blood. More specifically, the invention relates to hemoglobin-based oxygen carriers (HBOCs) for administration to mammals as a blood substitute or supplement, and processes for their preparation.
Hemoglobin, as the natural oxygen transporter component of blood, is an obvious candidate to form the basis of a blood substitute, e.g. as an aqueous solution. Extensive scientific work has been done and reported, on attempts to provide a satisfactory hemoglobin solution to act as a blood substitute. The chemical properties of hemoglobin outside the red blood cells are, however, markedly different from its properties inside the red blood cells, e.g. as regards its oxygen affinity. The need for some form of chemical modification of hemoglobin to render it suitable for use as a blood substitute has long been recognized and has been quite extensively investigated.
It is well known that hemoglobin comprises a tetramer of four sub-units, namely two a sub-units each having a globin peptide chain and two xcex2 sub-units each having a globin peptide chain. The tetramer has a molecular weight of approximately 64 kilodaltons, and each sub-unit has approximately the same molecular weight. The tetrameric hemoglobin in dilute aqueous solution readily dissociates into xcex1-xcex2 dimers and even further under some conditions to xcex1-sub-unit monomers and xcex2-sub-unit monomers. The dimers and monomers have too low a molecular weight for retention in the circulatory system of the body, and are filtered by the kidneys for excretion with the urine. This results in an unacceptably short half life of such a product in the body. The benefit of chemical bonding between the sub-units to ensure the maintenance of the tetrameric form (xe2x80x9cintramolecular cross-linkingxe2x80x9d) has previously been recognized. Also, the linking together of two or more tetrameric units to form hemoglobin oligomers and polymers of molecular weight greater than 64 kilodaltons (xe2x80x9cinter-molecular cross-linkingxe2x80x9d) has also been recognized as desirable in many instances.
Accordingly, one approach to developing HBOCs for clinical use has been intramolecularly cross-linking the hemoglobin units into stabilized tetramers, of molecular weight c. 64 kilodaltons, and optionally oligomerizing these tetramters into oligomers of 2-6 such tetramers, by intermolecular cross-linking. A variety of cross-linking reagents have been proposed for this purpose, including oxidatively ring-opened saccharides such as o-raffinose (U.S. Pat. No. 4,857,636 Hsia and U.S. Pat. No. 5,532,352 Pliura et al., for example), bifunctional imidates such as diethyl-malonimidate hydrochloride (U.S. Pat. No. 3,945,344 Muzur), halogenated triazines, divinylsulphones, diisocyanates, glutaraldehyde and other dialdehydes (U.S. Pat. No. 4,001,200 Bonsen et al.), bis-diaspirin esters (U.S. Pat. No. 5,529,719 Tye), bis- and tris-acyl phosphates (U.S. Pat. No. 5,250,665 Kluger et al.) and others
Another approach to the preparation of HBOCs with appropriate molecular weight for clinical use has been the coupling of hemoglobin to a biocompatible polysaccharide. Such conjugates would have the advantage as compared with cross-linked and oligomerized hemoglobins of requiring lower quantities of hemoglobin per unit of HBOC, and hence would be more economical to prepare, and have diminished hemoglobin-related toxicities. Conjugation of a colloid to hemoglobin in preparing an HBOC also permits control of fluid properties such as viscosity and colloid osmotic pressure by adjusting the size of the colloid, its degree of modification and the colloid-to-hemoglobin ratio. These same parameters can be used to control :he final molecular weight and vascular retention time of the product.
U.S. Pat. No. 4,064,118 Wong proposes the preparation of a blood substitute or blood extender by chemically coupling hemoglobin with a polysaccharide material selected from dextran and hydroxyethyl starch of molecular weight from about 5 kDa -2,000 kDa. Only the use of dextran is exemplified in this patent, however.
Baldwin et al. xe2x80x9cTetrahedronxe2x80x9d 37, pp 1723-1726 (1991) xe2x80x9cSynthesis of Polymer-Bound Hemoglobin Samplesxe2x80x9d describe the chemical modification of dextran and hydroxyethyl starch (HES) to form aldehyde-substituted polymers, and their subsequent reaction with hemoglobin, to form soluble, polymer-bound hemoglobin. Whilst the products so formed were capable of binding oxygen, they are reported as unsuitable for use as blood substitutes, since their oxygen-binding curves were considerably left-shifted, indicating that they have too high an oxygen affinity (P50 too low).
It is an object of the present invention to provide a novel HBOC.
It is a further object of the invention to provide a novel polysaccharide-hemoglobin conjugate useful as an HBOC.
It is a further object to provide a process for preparing a novel polysaccharide-hemoglobin conjugate useful as an HBOC.
In the process of the present invention, a polysaccharide is used, in oxidatively ring-opened form. In this oxidative form, at least a portion of the saccharide monomeric units are oxidized to present aldehyde groups. The oxidized polysaccharide so formed is then reacted with extracellular hemoglobin, so that the hemoglobin, through primary amine groups of the globin chains reacting with the aldehyde groups of the oxidized polysaccharide, covalently binds to the polysaccharide through Schiff base linkages. Initially and very rapidly there is formed a product which includes species of very high molecular weight, of the order of 500 kDa or higher, in substantial amounts and a wide molecular weight distribution (128xe2x86x92500 kDa).
On maintaining this product under appropriate conditions, in aqueous solution, it can be transformed, to a controlled extent, over a relatively short period of time (e.g 4-48 hours depending upon the conditions) to a much lower molecular weight product (90-200 kDa) with a much narrower molecular weight distribution. This product, after chemical reduction to reduce the Schiff base linkages between the hemoglobin and the polysaccharide to secondary amine bond, turns out to have properties such as oxygen affinity in the range P50=4 to 50 mmHg at 37xc2x0 C., depending on the ligand state of the hemoglobin at the time of conjugation, which makes it eminently suitable as a candidate for a hemoglobin based oxygen carrier for clinical use in mammals. The degree of transformation can be controlled by the timing of the application of the reduction step. Moreover, the resulting product contains no detectable unreacted hemoglobin which, if present, would dissociate to give xcex1xcex2-dimers suspected of causing renal injury, and no detectable amounts of excessively high molecular weight products (over about 500-600 kDa).
Thus according to the first aspect of the present invention, there is provided a polysaccharide-hemoglobin conjugate useful as a hemoglobin based oxygen carrier and having an oxygen affinity, expressed as partial pressure of oxygen environment required to maintain 50% oxygen saturation, P50 of =4-50 mmHg, at 37xc2x0 C., and containing no detectable residual unbound hemoglobin and no detectable residual amounts of components of molecular weight higher than about 500 kDa, said conjugate having been prepared by reacting hemoglobin with oxidized polysaccharide to form a high molecular weight conjugate complex, and allowing the high molecular weight conjugate complex to degrade by storage in solution at a suitable pH value, readily determinable by simple, routine experiments, and at a temperature from 2xc2x0 C. to about 45xc2x0 C. to form said polysaccharide-hemoglobin conjugate.
A further aspect of the invention provides a polysaccharide-hemoglobin conjugate useful as an oxygen transporter, comprising hemoglobin covalently linked through secondary amine linkages from amino groups on the hemoglobin to residues of aldehyde groups on the polysaccharide, said aldehyde groups having been formed by oxidative ring-opening of saccharide monomeric units of the polysaccharide.
According to another aspect, the present invention provides a process of preparing a hemoglobin based oxygen carrier which comprises reacting an oxidatively ring-opened polysaccharide carrying aldehyde groups with hemoglobin to form a Schiff based-linked conjugate thereof, allowing the conjugate to stand under conditions which effect molecular weight reduction of the conjugate, stabilizing the conjugate by reduction of the Schiff base linkages to stable, secondary amine linkages, and recovering a solution of the polysaccharide-hemoglobin conjugate so formed which has no detectable unbound hemoglobin residue and no detectable product residue of molecular weight greater than about 500-600 kDa,